Molecular Cell

Article

Distinct Roles for the XPB/p52 and XPD/p44Subcomplexes of TFIIH in Damaged DNAOpening during Nucleotide Excision RepairFrederic Coin,1,* Valentyn Oksenych,1 and Jean-Marc Egly1,*1 Institut de Genetique et de Biologie Moleculaire et Cellulaire, CNRS (UMR7104)/INSERM (U596)/ULP, BP 163,

67404 Illkirch Cedex, C.U. Strasbourg, France

*Correspondence: fredr@igbmc.u-strasbg.fr (F.C.), egly@igbmc.u-strasbg.fr (J.-M.E.)DOI 10.1016/j.molcel.2007.03.009

SUMMARY

Mutations in XPB, an essential subunit of thetranscription/repair factor TFIIH, lead to nucleo-tide excision repair (NER) defects and xero-derma pigmentosum (XP). The role of XPB inNER and the molecular mechanisms resultingin XP are poorly understood. Here, we show thatthe p52 subunit of TFIIH interacts with XPB andstimulates its ATPase activity. A mutation foundamong XP-B patients (F99S) weakens this inter-action and the resulting ATPase stimulation,thereby explaining the defect in the damagedDNA opening. We next found that mutations inthe helicase motifs III (T469A) and VI (Q638A)that inhibit XPB helicase activity preserve theNER function of TFIIH. Our results suggesta mechanism in which the helicase activity ofXPB is not used for the opening and repair ofdamaged DNA, which is instead only drivenby its ATPase activity, in combination with thehelicase activity of XPD.

INTRODUCTION

The human transcription/repair factor IIH (TFIIH) consists

of ten subunits. XPB, XPD, p62, p52, p44, p34, and p8/

TTDA form the core complex, while cdk7, MAT1, and cy-

clin H form the cdk-activating kinase (CAK) subcomplex,

linked to the core via XPD. Hereditary mutations in either

XPB, XPD, or p8/TTDA yield the xeroderma pigmentosum

(XP), XP combined with Cockayne syndrome (XP/CS), or

trichothiodystrophy (TTD) syndromes (Lehmann, 2003;

Giglia-Mari et al., 2004; Oh et al., 2006). These diseases

exhibit a broad spectrum of clinical features including

photosensitivity of the skin due to defects in nucleotide

excision repair (NER) (Lehmann, 2003). NER is part of a

cellular defense system that protects genome integrity

by removing a wide diversity of helix-distorting DNA le-

sions induced by ultraviolet (UV) light and bulky chemical

adducts. The removal of lesions requires their recognition

Mo

by the repair factor XPC-HR23b and the subsequent un-

winding of the DNA duplex by TFIIH. The single-stranded

structure is then stabilized by XPA and RPA, and the mar-

gins of the resulting DNA bubble are recognized by XPG

and ERCC1-XPF, thereby generating 30 and 50 incisions

relative to the damage (O’Donnovan et al., 1994; Sijbers

et al., 1996).

DNA helicases are motor proteins that can transiently

catalyze the unwinding of the stable duplex DNA mole-

cules using NTP hydrolysis as the source of energy. They

are characterized by seven ‘‘helicase motifs,’’ constituted

of conserved amino acid sequences (Tuteja and Tuteja,

2004). It was always hypothesized that XPB and XPD

helicase subunits of TFIIH supply opposite unwinding ca-

pacities required for local helix opening to form the open

DNA intermediates in NER (Bootsma and Hoeijmakers,

1993). Indeed, mutations in the ATP binding site of these

proteins inhibit NER in vivo and in vitro (Guzder et al.,

1994; Sung et al., 1988), due to a defect in the opening

of the damaged DNA structure (Coin et al., 2006). How-

ever, these studies used mutants that act by targeting

the ATPase A Walker I motif, but not the other helicase

motifs (from II to VI). Thus, questions remain as to whether

DNA opening during repair requires both XPB and XPD

helicases to open a short sequence of 24/32 nucleotides

encompassing the lesion.

Thus far, investigations of the mechanistic defects lead-

ing to XP, CS, or TTD have been beneficial in understand-

ing the function of XPB, XPD, and p8/TTDA in NER and

in transcription (Evans et al., 1997; Keriel et al., 2002;

Dubaele et al., 2003, Coin et al., 2006). In this study, we

unveiled the role of both the XPB and XPD subunits of

TFIIH in NER by analyzing several mutations found in XP

patients or other engineered mutations introduced in

highly conserved domains of the corresponding proteins.

We found that the helicase activity of XPB was not used for

damaged DNA opening, which is instead driven by its

ATPase activity, in combination with the helicase activity

of XPD. Furthermore, we demonstrated that the p52 sub-

unit of TFIIH upregulates the ATPase activity of XPB

through a direct XPB/p52 contact that is impaired in

XP-B patients. The TFIIH from these patient is unable to

induce the opening of the DNA around the lesion, due to

the incorrect XPB/p52 interaction and ATPase stimulation.

lecular Cell 26, 245–256, April 27, 2007 ª2007 Elsevier Inc. 245

Molecular Cell

Role of XPB and XPD in DNA Repair

RESULTS

The F99S Mutation in XPB Impairs Damaged

DNA Opening

To provide insights into the role of XPB in NER, we investi-

gated the DNA repair activity of two TFIIH complexes

purified from cell extracts of XP-B patients carrying either

the F99S (XP) or the T119P (TTD) mutations (Oh et al.,

2006) (Figure 1A, left panel). Western blot analysis reveals

a similar subunit composition of the immunopurified TFIIH/

XPB(WT), TFIIH/XPB(F99S), and TFIIH/XPB(T119P) com-

plexes (Figure 1A, right panel). Upon addition of TFIIH/

XPB(F99S) to a reconstituted in vitro dual incision assay

(Coin et al., 2004), a low level (10% activity) of excised

damaged oligonucleotides was observed, compared

with TFIIH/XPB(WT) (Figure 1B, NER, compare lanes 5

and 6 with lanes 3 and 4). TFIIH/XPB(F99S) was more effi-

cient in a reconstituted transcription assay (Gerard et al.,

1991) (75% activity) than in dual incision (Tx, compare

lanes 5 and 6 with lanes 3 and 4). The T119P mutation

did not affect either dual incision or transcription activities

(compare lanes 7 and 8 with lanes 3 and 4).

Given the role of TFIIH in NER, we carried out a perman-

ganate footprinting assay measuring the opening of the

DNA around the damage (Evans et al., 1997). Addition of

either TFIIH/XPB(WT) or TFIIH/XPB(T119P) to a reaction

containing XPC-HR23b, in addition to the cisplatinated

DNA fragment, resulted in an increased sensitivity of nu-

cleotides at positions T-4, T-5, and, to a lesser extent,

T-7 and T-10 indicative of DNA opening (Figure 1C, com-

pare lane 2 with lanes 5 and 9). In contrast, addition of

TFIIH/XPB(F99S) did not trigger a detectable opening of

the damaged DNA (Figure 1C, lane 7). However, further

addition of the NER factor XPA to TFIIH/XPB(F99S) pro-

moted a weak but significant opening of the DNA, com-

pared with the full opening obtained with either TFIIH/

XPB(WT) or (T119P) (Figure 1C, compare lane 8 to lanes

6 and 10). This defect in DNA opening parallels and ex-

plains the low removal of damaged oligonucleotides

seen in Figure 1B.

To dissect the molecular mechanism of the NER defect

observed with the F99S mutation, we purified from bacu-

lovirus-infected insect cells a recombinant TFIIH complex

(IIH6) containing the six subunits of the core TFIIH (XPB,

XPD, p62, p52, p44, and p34). The following experiments

were performed only with the core TFIIH, since the CAK

complex did not play any role in our in vitro NER assay

(Coin et al., 2006). Similarly to the endogenous TFIIH/

XPB(F99S), the recombinant IIH6/XPB(F99S) showed a

lower repair activity, compared with either IIH6/XPB(WT)

or (T119P) (Figure 1D, compare lane 5 with lanes 1 and

7). Interestingly, the addition of the NER-specific TFIIH

subunit p8/TTDA to IIH6/XPB(F99S) did not stimulate inci-

sion, compared with the increase in the removal of dam-

aged oligonucleotides observed with either the IIH6/

XPB(WT) or IIH6/XPB(T119P) complexes (Figure 1D,

compare lane 6 with lanes 2 and 8). Next, we observed

that addition of p8/TTDA and XPA to IIH6/XPB(F99S) did

246 Molecular Cell 26, 245–256, April 27, 2007 ª2007 Elsevier In

not trigger optimal opening of the damaged DNA in a per-

manganate footprinting assay, compared with either IIH6/

XPB(WT) or IIH6/XPB(T119P) (Figure 1E, compare lanes

10–12 with lanes 4–6 and 13–15). As a control, mutation

in the XPB ATPase A Walker I motif totally abolished the

IIH6/XPB(K346R) repair activity (Figure 1D, lane 3), due

to an inhibition of the damaged DNA opening (Figure 1E,

lane 7) and regardless of the presence of p8/TTDA (Fig-

ure 1D, lane 4, and Figure 1E, lanes 8 and 9).

Finally, the recruitment of both TFIIH and XPA to the

lesion was tested in vivo following local UV irradiation of

wild-type MRC5 and XPCS2BA (bearing the F99S muta-

tion) nuclei (Volker et al., 2001). Fluorescence signals of

XPB colocalized with cyclobutane pyrimidine dimer (CPD)

spots both in wild-type MRC5 and XPCS2BA cells (Fig-

ures 2A–2D), indicating that TFIIH/XPB(F99S) translocates

to the sites of DNA photolesions. In contrast, XPA was not

recruited to the lesions in XPCS2BA, compared to MRC5

cells (Figures 2E–2H). At this point, we concluded that the

repair defect harbored by TFIIH/XPB(F99S) is at the open-

ing step, following the binding of TFIIH to the damaged

DNA.

p52 Stimulates the ATPase Activity of XPB

Having observed that the F99S mutation does not impair

the helicase activity of the recombinant XPB protein (data

not shown), we focused on the ATPase activity of XPB. We

observed that the core IIH6/XPB(F99S) complex displayed

a lower ATPase activity (30% activity) than those of IIH6/

XPB(WT) and IIH6/XPB(T119P) (Figure 3A). Enigmatically,

the free XPB(F99S) polypeptide exhibited a catalytic

ATPase activity similar to those of XPB(WT) or XPB(T119P)

(Figure 3B). These observations prompted us to examine

if XPB-interacting subunits in TFIIH could modulate its

ATPase activity. Addition of increasing amounts of p52, a

partner of XPB in TFIIH (Jawhari et al., 2002), to a fixed

amount of purified XPB significantly stimulated its ATPase

activity (Figure 3C, lanes 2–4). To the contrary, addition of

either p44 or p8/TTDA, two subunits of TFIIH that do not

interact with XPB, had no effect on the ATPase (Figure 3C,

lanes 5, 6, 8, and 9).

We next investigated if XPB(F99S) and XPB(T119P) were

detrimental for the XPB/p52 interaction. Equal amounts

of recombinant XPB(WT), XPB(F99S), and XPB(T119P),

immobilized on agarose beads, were incubated with p52-

expressing extracts. Following extensive washing, we

observed in our experimental conditions that XPB(F99S)

interacts much less with p52 than do XPB(WT) or

XPB(T119P) (Figure 3D, compare lanes 8 and 9 and 5

and 6 with lanes 2 and 3). When tested in an ATPase

assay, p52 weakly stimulated XPB(F99S), compared with

XPB(WT) (Figure 3E, compare lanes 5–7 with lanes 2–4).

Altogether, these results demonstrate first that p52 regu-

lates XPB ATPase activity and second that a mutation

found in XP-B/CS patients weakens the interaction be-

tween the regulatory subunit p52 and XPB, leading to a

low stimulation of the ATPase activity and a reduced open-

ing of DNA around the damage.

c.

Molecular Cell

Role of XPB and XPD in DNA Repair

Figure 1. The F99S Mutation Impairs Damaged DNA Opening

(A) (Left) Schematic representation of XPB. The dark gray boxes indicate the helicase domains. The light gray box indicates the conserved N-terminal

domain. Mutations found in XP-B patients (F99S and T119P) and mutation in the ATPase A Walker I motif (K346R) are depicted. (Right) Two estab-

lished clones derived from the XPCS2BA cell line (mutation F99S) and expressing either the F99S (XP) or T119P (TTD) XPB (Riou et al., 1999) were

used together with the MRC5 control cell line for TFIIH purification. TFIIH/XPB(WT), TFIIH/XPB(F99S), and TFIIH/XPB(T119P) were immunoprecipi-

tated with antibody toward p44, a subunit of the core TFIIH, from whole-cell extracts and eluted with a competitor peptide (Coin et al., 1999). The

samples were resolved by SDS-PAGE and western blotted (WB) with anti-TFIIH antibodies. The subunits of TFIIH are indicated.

(B) Fifty and one hundred nanograms of TFIIH/XPB(WT) (lanes 3 and 4), TFIIH/XPB(F99S) (lanes 5 and 6), or TFIIH/XPB(T119P) (lanes 7 and 8) were

tested in a dual incision assay (NER) containing the recombinant XPC-HR23b, XPA, RPA, XPG, ERCC1-XPF factors and a closed-circular plasmid

containing a single 1,3-intrastrand d(GpTpG) cisplatin-DNA crosslink (Pt-DNA) as a template (Frit et al., 2002) or in a reconstituted transcription assay

(Tx) composed of recombinant TFIIB, TFIIF, TBP, TFIIE factors, the purified RNA polymerase II, and the adenovirus major late promoter template

(Gerard et al., 1991). Sizes of the incision products or transcripts are indicated.

(C) TFIIH (100 ng) was incubated with a radiolabeled linear DNA fragment from the Pt-DNA plasmid and 40 ng of XPC-HR32b. XPA (25 ng) was added

when indicated. Lane 1, Pt-DNA with BSA only. Residues are numbered with the central thymine of the crosslinked GTG sequence designated T0.

Arrows indicate KMnO4-sensitive sites. Adducted strand residues to the 30 and 50 ends of T0 are denoted by positive and negative integers (+N,�N).

(D) The recombinant IIH6/XPB(WT), IIH6/XPB(K346R) (mutated in the ATPase A Walker I site), IIH6/XPB(F99S), and IIH6/XPB(T119P) lacking CAK and

p8/TTDA were produced in baculovirus-infected insect cells (Tirode et al., 1999). TFIIH (100 ng) was tested in dual incision in the presence of 3 ng of

recombinant p8/TTDA when indicated (lanes 2, 4, 6, and 8).

(E) A KMnO4 assay was performed as described in Figure 1C with 100 ng of recombinant IIH6 complex incubated with a radiolabeled linear DNA

fragment from the Pt-DNA plasmid and XPC-HR32b. XPA (25 ng) and p8/TTDA (6 ng) were added when indicated. Lane 1, Pt-DNA with BSA only.

Lane 2, positive control with TFIIH purified from HeLa.

Molecular Cell 26, 245–256, April 27, 2007 ª2007 Elsevier Inc. 247

Molecular Cell

Role of XPB and XPD in DNA Repair

Figure 2. Recruitment of TFIIH and XPA at Sites of UV Damage

XPCS2BA(F99S) and wild-type MRC5 (labeled with blue beads) cells were plated on the same slide. Cells were UV irradiated with 70 J/m2 through a 3

mm pore filter and fixed 30 min later. Immunofluorescent labeling was performed using a rabbit polyclonal anti-XPB (A), a mouse monoclonal anti-CPD

(B and F) or a rabbit polyclonal anti-XPA (E). Nuclei were counterstained with DAPI (C and G), and slides were merged (D and H).

To map the region of p52 that is involved in the stimula-

tion of XPB ATPase activity, we designed the p52(1–304)

and the p52(305–462) truncated polypeptides (Figure 4A),

knowing that p52 interacts with XPB through two distinct

domains comprising the residues 1–135 and 304–381 (Ja-

whari et al., 2002). Equal amounts of purified recombinant

p52(WT), p52(1–304), and p52(305–462) were incubated

with fixed amount of purified recombinant XPB(WT) in an

ATPase assay. Both p52(WT) and p52(305–462) stimu-

lated XPB ATPase activity (Figure 4B, lanes 3–5 and 9–11,

respectively), while addition of p52(1–305) did not show

any significant effect (lanes 6–8). We also noticed that

p52(1–358), a truncated p52 polypeptide mimicking a

mutation found in yeast (Jawhari et al., 2002), efficiently

stimulated the XPB ATPase (data not shown and Jawhari

et al. [2002]). Altogether, our data indicate that the XPB

ATPase stimulation depends on the second XPB-interact-

ing domain in p52, delimited by residues 305 and 358.

Mutations in Helicase Domains of XPB Preserve

TFIIH Repair Activity

We next explored the combined action, if any, of both the

ATPase and helicase activities of XPB in NER. Since the

helicase activity of XPB depends on the integrity of seven

conserved motifs (Weeda et al., 1990), we designed two

recombinant XPB proteins. The first T469A mutation is

located in the helicase motif III, which is involved in the

unwinding of the DNA. Such mutation in the domain III

has been reported to impair the helicase activity of several

SF2 helicase family members (Pause and Sonenberg,

1992; Papanikou et al., 2004), including XPB (Lin et al.,

2005). The second Q638A mutation is located in the heli-

248 Molecular Cell 26, 245–256, April 27, 2007 ª2007 Elsevie

case motif VI, involved in the interaction with the single-

stranded DNA (Tuteja and Tuteja, 2004) (Figure 5A), and

was shown to be detrimental for XPB helicase activity

(Lin et al., 2005). We found that both recombinant

XPB(T469A) and (Q638A) displayed a very low 30–50 heli-

case activity, compared with XPB(WT) (Figure 5B, upper

panel, compare lanes 5 and 6 and 8 and 9 with lanes 2

and 3), while neither T469A nor Q638A mutations inter-

fered with XPB ATPase activity (Figure 5B, lower panel).

Remarkably, IIH6/XPB(T469A) and IIH6/XPB(Q638A) re-

moved damaged DNA as efficiently as did IIH6/XPB(WT)

in a dual incision assay (Figure 5C, compare lanes 7–9

and 10–12 with lanes 1–3), while their ability to allow RNA

synthesis was decreased when added to a reconstituted

transcription system (50% and 20% activity, respectively)

(Figure 5D, compare lanes 7–9 and 10–12 with lanes 1–3).

In contrast, IIH6/XPB(K346R), deficient in the ATPase

activity of XPB, was inactive both in DNA repair and tran-

scription (Figures 5C and 5D, lanes 4–6). In a permanga-

nate assay, addition of either TFIIH/XPB(WT) or TFIIH/

XPB(T469A) to a reaction containing XPC-HR23b and

p8/TTDA resulted in a DNA opening around the lesion, de-

pendent on the addition of ATP (Figure 5E, lanes 3–5 and

9–11). By contrast, a mutation in the ATPase A Walker

motif I (TFIIH/XPB[K346R]) inhibited the DNA-damaged

opening (lanes 6–8) (Coin et al., 2006).

To assess the importance of the helicase activity of XPB

during NER in vivo, a host cell reactivation assay was per-

formed (Carreau et al., 1995). A reporter construct (pLuc),

carrying a luciferase gene, was damaged by UV irradiation

and transfected in the repair-deficient CHO27-1 cells,

mutated in the XPB (Ma et al., 1994), together with an

r Inc.

Molecular Cell

Role of XPB and XPD in DNA Repair

undamaged control vector coding for b-galactosidase and

an expression vector coding for the human XPB proteins

of interest. Expression of the UV-irradiated reporter gene

was suppressed in CHO27-1, due to their repair defect (Fig-

ure 5F, compare lane 2 with lane 3). Cotransfection of

XPB(WT)cDNApartially restored luciferase gene expression

(lanes 3 and 4), while cotransfection of XPB(fs740) cDNA

containing a mutation that abolishes NER (Coin et al.,

2004) did not (Figure 5F, lane 6). The recovery of luciferase

activity is incomplete, probably due to species-specific

differences between human and hamster XPB. Cotrans-

fection of XPB(T469A) cDNA allowed an increase in the lu-

ciferase expression that reaches the level observed with

XPB(WT) (lanes 4 and 5), demonstrating that the T469A

mutation spares TFIIH repair activity in vivo. Altogether,

we show that, while the helicase activity of XPB is dispens-

able for effective NER, its ATPase activity is required.

Mutations Impairing XPD Helicase Activity Thwart

the Repair Activity of TFIIH

We next addressed if XPD, the other helicase of TFIIH,

might be contributing to the opening of the damaged DNA.

We designed recombinant IIH6 complexes with XPD con-

taining either the R658H, R683W, or R722W mutations

found within XP/TTD patients or the K48R mutation lo-

cated in the ATPase A Walker I motif (Figure 6A). As they

prevent the interaction of XPD with p44, the R683W and

R722W mutations impair the helicase activity of XPD,

while R658H conveys to its partial inhibition (Dubaele

et al., 2003). The K48R mutation inhibits both ATPase

and helicase activities of XPD (Tirode et al., 1999). Using

the permanganate footprinting assay, we showed that

damaged DNA opening was impeded in the absence of

the XPD ATPase activity (Figure 6B, lanes 5 and 6). Simi-

larly, R683W and R722W hindered damaged DNA open-

ing, even in the presence of p8/TTDA (Figure 6B, lanes

7, 8, 11, and 12). In contrast, R658H is sensitive to the

addition of p8/TTDA, and we observed a limited but signif-

icant opening of the DNA around the lesion with the corre-

sponding mutated complex (Figure 6B, lane 10). Interest-

ingly, the rate of dual incision activity obtained with the

IIH6/XPD(R658H) complex (50% activity) (Figure 6C,

compare lanes 6–8 with lanes 3–5) parallels the level of

DNA opening. Finally, the presence of the XPB(T469A)

subunit within IIH6/XPD(R658H) resulted in the IIH6/

XPD(R658H)/XPB(T469A) complex exhibiting a dual inci-

sion activity similar to that of IIH6/XPD(R658H), regardless

of the presence of p8/TTDA (Figure 6C, compare lanes 9–

11 with lanes 6–8). In conclusion, our results reveal that

DNA opening in NER depends on the ATPase, but not

on the helicase, activity of XPB in combination with the

helicase activity of XPD.

DISCUSSION

p52, a New Regulatory Subunit in TFIIH

By dissecting the repair defect induced by the F99S muta-

tion found in XP-B patients, we have shed light on the im-

Mol

portance of the partnership between XPB and p52 in NER.

We demonstrated that the F99S mutation in XPB weakens

the interaction with p52 and the resulting stimulation of its

ATPase activity, thereby inhibiting the opening of the dam-

aged DNA and the removal of the lesion. Given its role, p52

can be considered as a regulatory subunit of the ATPase

activity of XPB within TFIIH. Recently, it was shown that

p8/TTDA participates in the regulation of the ATPase ac-

tivity of XPB within the TFIIH complex, even though these

two subunits do not interact. However, p8/TTDA interacts

with p52 (Coin et al., 2006), and it is likely that the free p8/

TTDA, which was shown to shuttle between the cytoplasm

and nucleus and to associate with TFIIH when NER-spe-

cific DNA lesions are produced (Giglia-Mari et al., 2006),

would regulate or stabilize the XPB/p52 interaction within

the TFIIH complex. Thus, the binding of p8/TTDA to TFIIH

and the resulting stimulation of the XPB ATPase activity by

p52 might constitute a crucial NER checkpoint, deciding

whether or not a lesion will be removed. In the light of

the 3D structure of an archea XPB homolog (Fan et al.,

2006), it was proposed that ATP hydrolysis by XPB drives

a large conformational change inducing a reorientation

of a moiety of XPB and its wrapping around the DNA.

Accordingly, it is likely that p52 together with p8/TTDA

regulates this conformational change through the stimula-

tion of the ATPase activity of XPB.

Is XPB a Conventional Helicase in NER?

The removal of lesions depends on the opening of the DNA

around the damaged site. Natural mutations in either the

XPB or the XPD proteins can disable DNA opening (Evans

et al., 1997). A remaining question is this: do both DNA

helicase activities function during the NER reaction? Mu-

tations in the ATP binding site of XPB and XPD totally

impede the formation of the open DNA structure in NER

(Sung et al., 1988; Guzder et al., 1994; Coin et al., 2006).

However, such observations indicate that the hydrolysis

of ATP by XPB is essential for the function of TFIIH in repair

but do not demonstrate that the helicase activity of XPB is

required for NER. It raises the possibility that the ATPase

activity is not only a provider of energy for the helicase

action but also displays another independent and distinct

function. This hypothesis is strengthened by the fact that

the stimulation of the ATP hydrolysis by the XPB/p52 part-

nership does not increase XPB helicase activity (data not

shown). Furthermore, TFIIH-bearing mutations in the heli-

case motifs III or VI of XPB are still functional in NER. This

supports the idea that XPB doesn’t act as a conventional

helicase in NER, a role that is devoted to XPD, the other

helicase of TFIIH. Indeed, we demonstrated that muta-

tions weakening the contact of XPD with its p44 regulatory

subunit (Coin et al., 1998; Dubaele et al., 2003) impair

damaged DNA opening. In this context, we favor a model

in which the wrapping of XPB around the DNA will allow for

a local melting of the double-stranded DNA around the

lesion that would favor the correct anchoring of the XPD

helicase. XPB would therefore play the role of a wedge,

using ATP to keep the two strands of the DNA around

ecular Cell 26, 245–256, April 27, 2007 ª2007 Elsevier Inc. 249

Molecular Cell

Role of XPB and XPD in DNA Repair

250 Molecular Cell 26, 245–256, April 27, 2007 ª2007 Elsevier Inc.

Molecular Cell

Role of XPB and XPD in DNA Repair

Figure 4. Mapping the Domain of p52

Involved in the Stimulation of the XPB

ATPase

(A) Schematic representation of p52. The

stretches of highly conserved residues in

eukaryotes are indicated in black, and XPB

binding regions are delimited.

(B) Purified FLAG-tagged p52(WT) (25, 50, and

100 ng) (lanes 3–5), p52(1–304) (lanes 6–8), and

p52(305–462) (lanes 9–11) were incubated with

50 ng of recombinant XPB (lanes 2–11) and

then resolved by SDS-PAGE and western

blotted against XPB and the FLAG tag (WB)

or incubated in an ATPase assay (ATPase).

the lesion apart, allowing XPD to unwind the DNA. This

model also brings together the modes of action of XPB

in the opening of the DNA around the promoter and

around the lesion (Lin et al., 2005).

Molec

TFIIH Repair Disorders, a Matter of Interactions

So far in humans, viable TFIIH mutations have been found

only in XPB, XPD, and p8/TTDA. Our work reveals that mu-

tations found in XP-B and -D patients never affect the

Figure 3. The F99S Mutation Thwarts the Interaction between XPB and p52 and the Stimulation of XPB ATPase Activity

(A) Increasing amounts (50, 100, 200, and 400 ng) of either IIH6/XPB(WT), IIH6/XPB(F99S), or IIH6/XPB(T119P) were tested in an ATPase assay. The

graph represents the percentage of phosphate released (Pi/[ATP+Pi]) from three independent experiments.

(B) Increasing amounts (20, 40, 80, and 160 ng) of either XPB(WT), XPB(F99S), or XPB(T119P) were tested in an ATPase assay. The graph represents

the percentage of phosphate released (Pi/[ATP+Pi]) from three independent experiments.

(C) Purified XPB (50 ng) (lanes 2–9) was tested in an ATPase assay in the presence of 50 and 100 ng of purified p52 (lanes 3 and 4), 10 and 20 ng of

purified p8/TTDA (lanes 5 and 6), or 50 and 100 ng (lanes 8 and 9) of purified p44 subunits of TFIIH.

(D) XPB(WT) (lanes 1–3), XPB(F99S) (lanes 4–6), or XPB(T119P) (lanes 7–9) from baculovirus-infected insect cell extracts were immunoprecipitated

with anti-XPB antibody. Following washes, beads were incubated with baculovirus-infected insect cell extracts expressing p52, washed at 0.2 or

0.4 M KCl as indicated, and then resolved by SDS-PAGE and western blotted. HC, Ab heavy chain.

(E) XPB(WT) (lanes 2–4) or XPB(F99S) (lanes 5–7) from baculovirus-infected insect cell extracts were immunoprecipitated with anti-XPB antibody.

Following washes, beads were incubated with increasing amounts of baculovirus-infected insect cell extracts expressing p52, washed at 0.4 M

KCl, and then tested in an ATPase assay or resolved by SDS-PAGE and western blotted as indicated. HC, Ab heavy chain.

ular Cell 26, 245–256, April 27, 2007 ª2007 Elsevier Inc. 251

Molecular Cell

Role of XPB and XPD in DNA Repair

Figure 5. Mutations in Helicase Domains of XPB Spare TFIIH Repair Activity

(A) Schematic representation of XPB. Mutations introduced in conserved helicase domains are indicated.

(B) (Upper panel) Immunoprecipitated recombinant XPB (50 and 200 ng) (expressed in baculovirus-infected insect cells) were tested in helicase assay

using a bidirectional probe (Coin et al., 1998) at either 37�C or 4�C as indicated. Lane 1 contains highly purified TFIIH from HeLa cells. D, probe has

been heated 5 min at 100�C. Lane 11 is a control without XPB. Values under the autoradiograph represent the percentage of probe (30/50 ) released

relative to control lane 12. (Lower panel) Immunoprecipitated XPB (50 and 200 ng) was tested in an ATPase assay at 30�C or 4�C when indicated.

Values under the autoradiograph represent the percentage of phosphate released (Pi/[ATP+Pi]).

(C) One hundred nanograms of either IIH6/XPB(WT) (lanes 1–3), IIH6/XPB(K346R) (lanes 4–6), IIH6/XPB(T469A) (lanes 7–9), or IIH6/XPB(Q638A) (lanes

10–12) was tested in a dual incision assay in the presence of increasing amount of p8/TTDA (1.5 and 3 ng) as indicated.

(D) The TFIIH (50, 100, and 200 ng) tested in (C) was assessed in a reconstituted transcription assay as in Figure 1B, in the presence of 50 ng of

recombinant CAK complex (Rossignol et al., 1997). The size of the transcript is indicated.

(E) Fifty and one hundred nanograms of IIH6/XPB(WT) (50 and 100 ng) (lanes 3–5), IIH6/XPB(K346R) (lanes 6–8), or IIH6/XPB(T469A) (lanes 9–11) were

incubated with p8/TTDA and XPC-HR32b, with or without ATP, in a KMnO4 assay. Lane 1, Pt-DNA with BSA only. Lane 2, positive control with TFIIH

purified from HeLa.

(F) CHO27-1 cells were transfected with pLuc plasmid expressing the luciferase gene previously irradiated (lanes 3–6) or not (lane 2) in combination

with pcDNA expressing either XPB(WT) (lane 4), XPB(T469A) (lane 5), or XPB(fs740) (lane 6). Repair complementation was assessed by monitoring

252 Molecular Cell 26, 245–256, April 27, 2007 ª2007 Elsevier Inc.

Molecular Cell

Role of XPB and XPD in DNA Repair

Figure 6. The XPD Helicase Activity

Opens Damaged DNA in NER

(A) Schematic representation of XPD. The heli-

case domains are indicated by dark gray boxes.

The region of interaction with the regulatory

subunit p44 is highlighted (Coin et al., 1998;

Dubaele et al., 2003).

(B) A KMnO4 assay, as described in Figure 1C,

was performed with 100 ng of TFIIH incubated

with the Pt-DNA, XPC-HR32b, and XPA. Eight

nanograms of p8/TTDA was added when indi-

cated. Lane 1, Pt-DNA with BSA only.

(C) One hundred nanograms of IIH6/XPD(WT)

(lanes 3–5), IIH6/XPD(R658H) (lanes 6–8), or

IIH6/XPD(R658H)/XPB(T469A) (lanes 9–11)

was tested in a dual incision assay with increas-

ing amounts of p8/TTDA (1.5 and 3 ng). Values

under the autoradiograph represent the repair

activity (RA) calculated from three independent

experiments.

activity of the protein per se (helicase for XPD, ATPase for

XPB) but rather disturb the interactions of these enzymes

with their regulatory partners (p44 for XPD and p52 for

XPB), explaining how patients with such mutations may

exist. In this context, one could ask, why have no patients

with mutations in p52 been described yet? Interestingly,

point mutations in the Drosophila homolog of the human

p52, destabilizing the interaction between Dmp52 and

XPB and limiting the stimulation of the ATPase activity of

the latter, give rise to UV sensitivity with melanotic tumors

Mol

in larvae and pupae, and chromosome instability, all char-

acteristics of a DNA repair defect (Fregoso et al., 2007).

The study of such a fly model in association with a full

characterization of the molecular defects associated to

mutations found in humans will be useful to explain the

clinical jumble associated with XP, CS, and TTD patients

mutated in TFIIH genes.

Taken as a whole, our data show that some of the pre-

viously called ‘‘structural’’ subunits of TFIIH (i.e., p52, p44,

p62, and p34) (Tirode et al., 1999) do indeed have crucial

luciferase activity in cell lysates (48 hr posttranfection) normalized with the internal b-galactosidase standard. Results are expressed as relative

luciferase activity. The error bars were calculated on the basis of three independent experiments. Fifty micrograms of total extract was resolved

by SDS-PAGE and western blotted (WB) with mouse anti-human XPB antibody (Coin et al., 2004).

ecular Cell 26, 245–256, April 27, 2007 ª2007 Elsevier Inc. 253

Molecular Cell

Role of XPB and XPD in DNA Repair

functions inside the complex by regulating XPB and XPD

enzymatic functions. Future studies will specially focus

on posttranslational modifications of these regulatory

subunits that may fine-tune TFIIH enzymatic activities,

enabling this factor to participate in a remarkable variety

of processes.

EXPERIMENTAL PROCEDURES

Construction of Plasmids

The cDNAs encoding XPB or p52, p52(1–304), and p52(305–462) were

inserted at the BamHI/EcoRI sites of the FLAG-tagged pSK278 vector

(BD Biosciences) in fusion with the FLAG peptide (MTKDDDDKH). The

XPB mutants were obtained by site directed mutagenesis (QuikChange

Site-Directed Mutagenesis Kit, Stratagene). The resulting vectors were

recombined with baculovirus DNA (BaculoGold DNA, Pharmingen) in

Spodoptera frugiperda 9 (Sf9) cells. For hosT cell reactivation assay,

XPB was inserted into the pcDNA3(+) expression vector (Invitrogen).

Protein Purification

Recombinant wild-type or mutated TFIIH, CAK, and p8/TTDA proteins

were purified as described (Tirode et al., 1999). TFIIH from human cells

was purified using mouse monoclonal anti-p44 antibody linked to pro-

tein A Sepharose. Following washes, the proteins were eluted from the

resin with an excess of the corresponding competitor peptide (Coin

et al., 1999). The FLAG-tagged p52 and XPB proteins were purified

with the anti-FLAG M2 antibody agarose affinity gel (Sigma-Aldrich)

followed by elution with an excess of competitor peptide.

Pull-Down Assay

Wild-type or mutant recombinant XPB from baculovirus-infected Sf9

cell lysates were immunoprecipitated O/N at 4�C in buffer A (50 mM

Tris-HCl [pH 7.9], 20% glycerol, 0.1 mM EDTA, 0.5 mM DTT) contain-

ing KCl (0.2 M) with anti-XPB (1B3) antibody linked to protein A Sephar-

ose beads. Beads were then extensively washed with buffer A (0.4 M

KCl) and re-equilibrated in buffer A (0.2 M KCl). Beads were then incu-

bated with baculovirus-infected Sf9 cell lysates expressing p52 for 2 hr

at 4�C in buffer A (0.2 M KCl), washed extensively with buffer A (0.4 M

KCl), and re-equilibrated in buffer A (0.05 M KCl) before being tested in

western blot or ATPase assay.

DNA Substrates for NER Assays

DNA substrates and dual/single incision assays were performed as

described (Riedl et al., 2003).

Host Cell Reactivation Assay

The pGL3 vector expressing Photinus pyralis (firefly) luciferase was

purchased from Promega and the pCH110 vector expressing the b-

galactosidase from Invitrogen. The pGL3 vector was UV irradiated

(254 nm, 1000 J/m2) at a concentration of 1 mg/ml in 10 mM Tris-

HCl (pH 8.0) and 1 mM EDTA. CHO27-1 cells were transfected in a

6-well plate at a confluence of 95% using Lipofectamine Plus (Invitro-

gen). Each transfection mixture contained 500 ng of pGL3 (UV+/�),

100 ng of pCH110 (nonirradiated), and 10 ng of pcDNAXPB(WT),

(T469A) or (fs740). After 4 hr of incubation, the transfection reagents

were replaced by medium. Cells were lysed after 24 hr to measure lu-

ciferase activity on a microtiter plate luminometer (Dynex). All results

(mean values of at least five measurements) were normalized by calcu-

lating the ratios between luciferase and galactosidase activities.

KMnO4 Footprinting Assay

This assay has been described in Tapias et al. (2004). Briefly, the dam-

aged strand probe was obtained upon AgeI digestion of the Pt-DNA

and radiolabeling at the 30 end in a Klenow reaction, the Pt adduct be-

ing located at 156 bp from the labeled end. The resulting fragment was

purified by the ‘‘crush and soak’’ method after migration in a 5% non-

254 Molecular Cell 26, 245–256, April 27, 2007 ª2007 Elsevier I

denaturating PAGE. Reactions (75 ml) were carried out in 20 mM

HEPES/KOH (pH 7.6), 60 mM KCl, 5 mM MgCl2, 10% glycerol,

1 mM dithiothreitol, 0.3 mM EGTA, 1 mM ATP, 0.4% polyvinyl alcohol,

and 0.4% polyethylene glycol 10,000 buffer containing the labeled cis-

platinated probe (40 fmol) and, when indicated, the NER factors XPC/

HR23b (40 ng) and XPA (25 ng). After incubation at 30�C for 15 min, 3 ml

of 120 mM KMnO4 was added, and oxidation was allowed to proceed

for 3 min at room temperature before reduction by adding 6 ml of 14.6 M

b-mercaptoethanol for 5 min on ice. After organic extraction and eth-

anol precipitation, dried pellets were resuspended in 100 ml of a solu-

tion containing 1 M piperidine, 1 mM EDTA, and 1 mM EGTA and incu-

bated at 90�C for 25 min. Next, samples were ethanol precipitated, and

final pellets were recovered in 10 ml of loading buffer and analyzed in

8% urea-PAGE.

Helicase Assay

The helicase substrate was obtained by annealing 5 ng of an oligonu-

cleotide corresponding to the fragment 6219–6255 of single-stranded

M13mp18 (�) DNA to 1 mg of single-stranded M13mp18 (+). The result-

ing heteroduplex was digested for 1 hr at 37�C with EcoRI (New En-

gland Biolabs) and then extended to 21 and 20 bp, respectively, with

the Klenow fragment (5 units) in the presence of 50 mM dTTP and

7 mCi [a-32P]dATP (3000 Ci/ mmol, Amersham). Helicase assay was

then performed as described (Coin et al., 1998).

ATPase Assay

Protein fractions were incubated for 2 hr at 30�C in the presence of

1 mCi [g-32P]ATP (7000 Ci/mmol, ICN Pharmaceuticals) in a 20 ml reac-

tion volume in 20 mM Tris-HCl (pH 7.9), 4 mM MgCl2, 1 mM DTT,

50 mg/ml BSA, and, when indicated, 120 ng of supercoiled double-

strand DNA (pSK). Reactions were stopped by adding EDTA to

50 mM and SDS to 1% (w/w). The reactions were then diluted

5-fold, spotted onto polyethylenimine (PEI) TLC plates (Merck), run in

0.5 M LiCl/1 M formic acid, and autoradiographed.

Local UV Irradiation

The cells were rinsed with PBS and were covered with an isopore poly-

carbonate filter with pores of 3 mm diameter (Millipore, Bedford, MA).

Cells were then exposed to UV irradiation with a Philips TUV lamp

(predominantly 254 nm) at a dose of 70 J/m2. Subsequently, the filter

was removed, the medium was added back to the cells, and cells

were returned to culture conditions for 30 min.

Fluorescence and Confocal Microscopy

Fibroblasts were grown for 2 days with fluorescent latex beads (Fluo-

resbrite Carboxylate Microspheres, Polysciences), fixed in 3% para-

formaldehyde for 10 min at room temperature, and permeabilized

with PBS/0.5% Triton for 5 min. After washing with PBS-Tween

(0.05%), the slides were incubated for 1 hr with the indicated anti-

bodies. After extensive washing with PBS-Tween, they were incubated

for 1 hr with Cy3-conjugated goat anti-rabbit IgG (Jackson Laborato-

ries) or with anti-mouse Alexa 488 IgG (Jackson Laboratories) diluted

1:400 in PBS-Tween (0.5%). The slides were counterstained for DNA

with DAPI prepared in Vectashield mounting medium (Vector lab). All

images were collected using a Leica Confocal TCS 4D microscope

equipped with both UV laser and an Argon/Kripton laser and standard

filters to allow collection of the data at 488 and 568 nm. The software

TCSTK was used for three-color reconstructions, and figures were

generated using the PLCHTK software.

Antibodies

Mouse monoclonal antibodies toward TFIIH subunits were used as de-

scribed (Marinoni et al., 1997). Anti-FLAG M2 is from SIGMA. Primary

antibodies (the final dilutions are indicated in parentheses) used in fluo-

rescent labeling were rabbit IgG polyclonal anti-XPB (S-19, Santa Cruz

Biotechnology) (1:200), rabbit IgG polyclonal anti-XPA (1:200) (FL-273,

Santa Cruz Biotechnology), and mouse IgG monoclonal anti-CPD

nc.

Molecular Cell

Role of XPB and XPD in DNA Repair

(TDM2) (1:2000) (MBL International Corporation). Secondary anti-

bodies used in this study were Alexa 488 anti-mouse IgG and Cy3-

conjugated goat anti-rabbit IgG.

ACKNOWLEDGMENTS

We thank Mario Zurita for sharing results; Miria Stefanini, Tiziana

Nardo, and Olga Zlobinskaya for fruitful discussions; and Alain Sarasin

for the cell lines. We are grateful to Annabel Larnicol for excellent tech-

nical expertise. We thank Jean-Luc Weickert and Isabelle Kolb for their

invaluable assistance. We are grateful to Renier Velez-Cruz for critical

reading of the manuscript. This study was supported by funds from the

French League against Cancer (CDP 589111) and the French National

Research Agency (NR-05-MRAR-005-01). We also thank the French

Institute for Rare Diseases (number A03098MS). V.O is supported

by the EEC (LSHG-CT-2005-512113).

Received: October 24, 2006

Revised: February 19, 2007

Accepted: March 5, 2007

Published: April 26, 2007

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